A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

T he coronavirus disease 2019  pandemic, caused by the β-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global health emergency. Vaccines eliciting neutralizing antibodies against the Spike protein of SARS-CoV-2 have shown high effectiveness 1,2 . However, the level of neutralizing antibodies declines after vaccination 3 and at-risk patient groups, including transplant recipients, individuals suffering from B cell leukemia, and auto-immune disease patients on selected immunosuppressive regimens (such as B cell depleting reagents), exhibit lower humoral and cellular immunity after vaccination [4][5][6] . Moreover, the mutation rate of coronaviruses is substantial and certain mutations in the Spike protein of SARS-CoV-2 have emerged that can lead to a decline in neutralizing antibodymediated protection 7,8 . In order to address this clinical problem, a third vaccination for solid organ transplant recipients is becoming the standard of care in more and more countries, and supplementary T cell-focused strategies have been suggested 9,10 .
While boosters with the current vaccines likely lead to the generation of enhanced levels of neutralizing antibodies, it is uncertain if this will improve protection in the aforementioned risk groups and provide protection against the emergence of novel SARS-related viruses. However, booster vaccination may also lead to enhanced T cell responses, which is expected to contribute to the control of SARS-CoV-2 infection as evidenced by their association to disease severity and protection in humans [11][12][13][14][15][16] and animal models 17,18 . Moreover, T cell-focused vaccines can be directed to more conserved regions of the coronavirus, which may lead to a broad T cell-based cross-protection against multiple coronavirus strains and likely independently expands the protection provided by antibodies. Such T cellmediated protection is important given the potential emergence of similar viruses in the future that pose a significant threat to global public health. Previous studies showed that T cells can mediate protection by themselves against SARS-CoV-1 19,20 but whether T cell-eliciting vaccines can protect against SARS-CoV-2 is unclear. Here, we demonstrate that a peptide vaccine exclusively eliciting CD8 + T cell responses against a single epitope present in the Spike protein delivers full protection against SARS-CoV-2, provided that the vaccine was administered in a double booster regimen, leading to the induction of high numbers of circulating and tissue-resident memory T (T RM ) cells. A third vaccination was especially critical to achieve cytokine polyfunctionality and induction of elevated T RM cell numbers in the lungs and liver, and additionally fueled retrograde migration of T RM cells into the circulation. Thus, booster vaccinations eliciting strong CD8 + T cell responses are a promising strategy against coronavirus-mediated disease independent of neutralizing antibodies.

Results
Vaccines eliciting antibodies against confirmational proteins but not linear B cell epitopes are neutralizing and effective against SARS-CoV-2 infection. Single B and T cell epitope vaccines can provide effective approaches against a variety of pathogens and malignancies [21][22][23][24] . To investigate the efficacy of linear B cell epitope vaccines against SARS-CoV-2 infection, we selected five different linear epitopes, present at different locations in the Spike protein, which were considered to potentially able to elicit protective antibodies [25][26][27] . To test their immunogenicity, C57BL/6 mice were vaccinated with synthetic long peptide (SLP)based vaccines each containing a single B cell epitope. After three vaccinations, however, these SLP vaccines did not elicit Spikespecific antibody responses ( Supplementary Fig. 1a). To improve this synthetic vaccine, we tested the particular influence of adjuvants and of CD4 T cell help for the specific antibody induction of the B cell-SLP vaccines. To this end, the SLP-based vaccines were coupled with the universal helper epitope PADRE and adjuvanted with CpG and Incomplete Freund's Adjuvant (IFA). The combination of these adjuvants acted synergistically to elicit high levels of antibodies against the Spike protein (Fig. 1a). This synergistic effect correlated with a higher induction of PADRE-specific CD4 + T cell responses (Fig. 1b, c). Coupling of the PADRE epitope to the linear B cell epitopes, both N-terminal and C-terminal, also contributed to the improvement of the Spike-specific IgG antibody response, instead of combining the linear B cell epitopes and PADRE epitope in a single vaccine ( Fig. 1d and Supplementary Fig. 1b). Moreover, depletion of CD4 + T cells during the vaccination period resulted in a decreased Spike-specific antibody response ( Supplementary  Fig. 1c), highlighting the need from CD4 + T cells for an optimal IgG response. CD8 + T cell responses were not induced by the B cell-SLP vaccines ( Supplementary Fig. 1d).
To determine whether immunization with optimized linear B cell epitope vaccines enables protective immune responses against SARS-CoV-2, we used transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor under the regulation of the cytokeratin 18 (K18) promoter, which develop severe lung disease in response to SARS-CoV-2 infection 28 . The K18-hACE2 mice were vaccinated three times with a two week interval with either one CpG/IFA adjuvanted PADRE-coupled B cell-SLP vaccine or a combination of the five different B cell-SLP vaccines. Spike-specific IgG antibody responses were elicited and reached high titers after the third (3 rd ) vaccination (Fig. 1e). Four weeks after the 3 rd vaccination, the K18-hACE2 mice were challenged intranasally with a lethal dose of SARS-CoV-2 and body weight and clinical symptoms were measured daily as parameters of disease. Both unvaccinated mice and mice that were vaccinated three times with the B cell-SLPs succumbed to SARS-CoV-2 infection as evidenced by >20% reduction in bodyweight and a moribund condition (Fig. 1f, g), showing that SLP vaccination with these linear B cell epitopes was not able to protect against SARS-CoV-2 infection. This inability of protection correlated with the incapability of antibodies to neutralize virus cell entry (Fig. 1h). In contrast, a DNA vaccine encoding the entire Spike protein fully protected K18-hACE2 transgenic mice from SARS-CoV-2 infection, and this correlated with the ability to elicit neutralizing Spike-specific antibodies ( Fig. 1i-l). In addition, this DNA vaccine also induced a Spike-specific CD4 + and CD8 + T cell response ( Fig. 1m and Supplementary Fig. 1e). The response against an immunodominant CD8 + T cell epitope present in the Spike protein of SARS-CoV-1 and SARS-CoV-2 (i.e., Spike 539-546, VNFNFNGL) 29 increased upon the second (2 nd ) but not the 3 rd vaccination, and was characterized by an increased expression of the activation-associated NK cell receptor KLRG1 on the Spike 539-546 -specific CD8 + T cells (Fig. 1m, n). Overall, these results indicate that antibody responses to these single linear B cell epitopes are inferior for the induction of neutralizing antibodies, although we cannot exclude the possibility that single linear B cell epitopes exist that can elicit neutralizing antibodies. Nevertheless, antibody responses induced by vaccine platforms encoding properly folded proteins presenting conformational B cell epitopes are able to generate neutralizing antibodies and provide protection against SARS-CoV-2 infection.
A third vaccination with a peptide harboring a single T cell epitope protects against SARS-CoV-2 challenge. To determine whether an immune response induced by a single CD8 + T cell epitope vaccine was able to protect against SARS-CoV-2 infection, K18-hACE2 transgenic mice were vaccinated once, twice or three times with an SLP encoding the immunodominant CD8 + T cell epitope Spike 539-546 . Five weeks after the final vaccination with the Spike 539-546 -SLP, mice were intranasally challenged with a lethal dose of SARS-CoV-2 (Fig. 2a). SLP vaccination induced a Spike 539-546 -specific CD8 + T cell response in all mice, which increased with each booster immunization and remained elevated after contraction (Fig. 2b-d). CD4 + T cell responses and Spike-specific antibodies were not induced ( Supplementary Fig. 1f, g). Whereas 75% of the non-vaccinated mice succumbed to SARS-CoV-2 challenge, 66% and 50% of the mice that received one or two vaccinations, respectively, succumbed. Strikingly, all mice that received a 3 rd vaccine dose recovered from the SARS-CoV-2 challenge despite initial weight loss (Fig. 2e, f). Thus, a third vaccination with an SLP containing a single CD8 + T cell epitope protects against lethal SARS-CoV-2 infection.    Fig. 1 Vaccines eliciting antibodies against confirmational proteins but not linear B cell epitopes are neutralizing and effective against SARS-CoV-2 infection. a C57BL/6 mice were vaccinated subcutaneously (s.c.) on day 0, 14, and 28 with synthetic long peptides (SLPs) consisting of PADRE-coupled linear B cell epitopes adjuvanted or not with CpG and/or Inactivated Freund's Adjuvant (IFA) (Mock, IFA: n = 5; CpG, CpG+IFA: n = 3). Spike-specific IgG kinetics in blood are shown. b Representative flow cytometry plots of PADRE-specific CD4 + T cells in naïve unvaccinated mice and in day 21 postvaccinated mice. c PADRE-specific CD4 + T cell response on day 21 after vaccination as described in (a) (Naïve: n = 11; Mock, IFA: n = 5; CpG: n = 3; CpG+IFA: n = 13). *P = 0.048, ***P = 0.0007, ****P = <0.0001. d C57BL/6 mice were vaccinated as described in (a) with SLPs consisting of the B cell epitope (E) alone, the epitope combined (uncoupled) with the PADRE (P) peptide (E+P) or N-terminal and C-terminal PADRE coupled to the B cell epitope (P−E, E−P), and all adjuvanted with CpG and IFA (E, E+P, E−P: n = 5; P−E: n = 3). Endpoint dilutions of Spike-specific IgG in blood at day 42 after vaccination with Spike 405-422 -SLP are shown. *P = 0.035, **P = 0.005, ***P = 0.0004. e-h K18-hACE2 transgenic mice were vaccinated on day 0, 14, and 28 with one or five SLP vaccines containing a PADRE-coupled linear B cell epitope adjuvanted with CpG and IFA. Four weeks after the final vaccination, mice were intranasally infected with SARS-CoV-2 (e-g, Unvaccinated: n = 9; B cell SLP 1 epitope, B cell SLP 5 epitopes: n = 10). e Spike-specific IgG kinetics in blood. f Weight loss kinetics after SARS-CoV-2 challenge. g Survival graph after SARS-CoV-2 challenge. h SARS-CoV-2 neutralizing capacity by antibodies after B cell-SLP vaccination on day 42 measured by a WT virus neutralization assay (VNA) (Unvaccinated, B cell SLP 1 epitope, n = 2; B cell SLP 5 epitopes, n = 5). i-n K18-hACE2 mice were vaccinated intradermally on day 0, 21, and 42 with a Spike-encoding DNA vaccine. i Spike-specific IgG (Unvaccinated: n = 3; Spike DNA: n = 6), j weight loss kinetics and k survival graph after SARS-CoV-2 challenge (n = 10 per group). ****P = < 0.0001. l SARS-CoV-2 neutralizing capacity by antibodies after Spike DNA vaccination on day 55 measured by a VNA. Data represented as mean ± SEM (Unvaccinated: n = 1; Spike DNA: n = 6). m Kinetics of Spike 539-546 -specific CD8 + T cells in blood. Data represented as mean ± SEM (Unvaccinated: n = 3; Spike DNA: n = 6). n Kinetics of KLRG1 + CD62L − expression on Spike 539−546 -specific CD8 + T cells in blood. Data represented as mean ± SEM (n = 6).
Progressive differentiation of vaccine-specific CD8 + T cells after booster vaccination. To gain further insight into the differentiation of the vaccine-specific CD8 + T cells upon sequential vaccination, we studied these cells in-depth using detailed singlecell high dimensional clustering analysis and trajectory inference with an antibody panel targeting multiple markers of cellular activation and differentiation. Visualization based on the cluster distribution of the splenic Spike 539-546 -specific CD8 + T cells indicated that the cells after the 1 st vaccination were disparate from the 2 nd and 3 rd vaccination, while the 2 nd and 3 rd vaccination were largely overlapping (Fig. 4a). Strikingly, and consistent with the aforementioned data, a large KLRG1 + cluster (cluster #3) co-expressing Ly6C and lacking CD62L, was more profoundly present in the 2 nd and 3 rd vaccination group (Fig. 4a-e). This cluster of cells also expressed CD44 and the cell proliferation marker Ki-67, but lacked expression of EOMES, TCF1, CXCR3, the activation-associated CD43 1B11 isoform, and PD-1. Cluster #2, which had a similar expression except for KLRG1, was also increased in the 2 nd and 3 rd vaccination group compared to the 1 st vaccination group (Fig. 4b-e). Other significantly different clusters, which were relatively reduced after the 2 nd and 3 rd vaccination compared to the 1 st vaccination, were characterized by either high levels of CD62L and TCF1, and lacking markers such as KLRG1 and CD44 (cluster #1) or expressed intermediate differentiation phenotypes (cluster #7: Fig. 4b-e). The single-cell trajectory algorithm Wanderlust 30 predicted that the developmental path progressed from the CD62L + CD44 − TCF1 + subset to transitional subsets harboring markers that were upregulated and retained (CD44, Ki-67, and Ly6C) or are eventually downregulated (EOMES, CXCR3, CD43 1B11 ) (Fig. 4a, f, g). The initially expressed CD62L and TCF1 were downregulated whereas KLRG1 expression was acquired at a late-stage indicating a progressive effector-memory T (T EM ) cell differentiation program after booster vaccination (Fig. 4f, g).
Vaccine-specific conditions drive the differentiation of elicited CD8 + T cells. To examine whether vaccine-specific conditions or the epitope itself influence the differentiation of the elicited CD8 + T cells, we vaccinated mice with SLP vaccines containing the epitope GP 34-41 (AVYNFATC) derived from lymphocytic choriomeningitis virus (LCMV) in a similar manner ( Supplementary  Fig. 2a). As observed with the Spike 539-546 -SLP immunization, each subsequent vaccination with GP 34-41 -SLP increased the magnitude of the vaccine-specific CD8 + T cell response, and this response remained higher after contraction ( Supplementary  Fig. 2b-d). Moreover, also the GP 34-41 -specific CD8 + T cells in the spleen, liver, and lungs where significantly increased upon each vaccination. In the spleen this was caused by an increment of the circulating GP 34-41 -specific CD8 + T cells, in the liver by the CD69 + GP 34-41 -specific CD8 + T RM cells and in the lungs by an increase in both the circulating as well as the tissue-resident T cells (Supplementary Fig. 2e). In addition, these increments were also reflected by increased polyfunctional cytokine producing GP 34-41 -specific CD8 + T cells in the spleen, liver, and lungs ( Supplementary Fig. 2f).
vaccination showed a similar pattern: a large KLRG1 + Ly6C + Ki-67 + cluster profoundly present in the 2 nd and 3 rd vaccination group and less differentiated clusters (CD62L + TCF1 + KLRG1 − ) that were relatively more abundant after the 1 st vaccination ( Supplementary Fig. 3a-d).
Further insight into the phenotype of the vaccine-specific CD8 + T cells upon sequential vaccination, using single-cell high dimensional mass cytometry with a panel of antibodies targeting markers of cellular activation and differentiation 31 (Supplementary Fig. 4a and Supplementary Table S3), confirmed the disparate phenotype after the 1 st vaccination compared to the 2 nd and 3 rd vaccination ( Supplementary Fig. 4b). Principal component analysis (PCA) based on the cluster frequencies of the GP 34-41 -specific memory CD8 + T cells in the spleen, liver and lungs confirmed the clear distinction between clusters present in one-time vaccinated versus clusters in two or three times vaccinated mice (Supplementary Fig. 4c). In addition, dual tSNE analysis on all samples of the GP 34-41 -specific CD8 + T cells, visualizing the segregation based on vaccination-associated patterns, corroborated that the 2 nd and 3 rd vaccination-specific phenotypes had considerable overlaps (Supplementary Fig. 4d). A low Jensen-Shannon (JS) divergence distance was calculated when comparing antigen-specific CD8 + T cells induced upon 2 nd and 3 rd vaccinations, indicating a high similarity between cells induced by these vaccinations, whereas a higher JS distance was apparent when comparing 1 st to 2 nd vaccinations or 1 st to 3 rd vaccinations ( Supplementary Fig. 4e). Visualization and quantification of the particular marker expression on the GP 34-41specific CD8 + T cell population indicated that in the spleen and lungs, the percentage CD69 − CD62L − T EM -like cells expressing high levels of KLRG1, Ly6C, and CX3CR1, and moderate expression of NKG2A, Sca-1 and the ectonucleotidase CD39 associated to the 2 nd and 3 rd vaccination, while the CD69 − CD62L + subsets related to single-dose vaccination (Supplementary Fig. 4f±h). The phenotype of CD69 + GP 34-41 -specific CD8 + T RM cells in the liver was slightly affected upon the 3 rd vaccination, and resulted in reduction of CD11c + CD122 + PD-1 − T RM cells and an increase in CD11c + CD122 − PD-1 + T RM cells (Supplementary Fig. 4h). Based on the induction of a highly similar phenotype by the Spike 539-546 -SLP and GP 34-41 -SLP vaccines, we conclude that the differentiation of the vaccine-elicited CD8 + T cells depends on (repeated) vaccinespecific conditions rather than the antigens itself.
A third vaccination triggers the remigration of T RM cells into the circulation. To fate map the T RM cell progeny upon booster vaccination, we used a reporter system based on the T RMrestricted transcription factor Hobit to evaluate the development of the T RM cells 32 . Here, Hobit lineage tracer (LT) mice, which were generated by crossing CD45.1 Hobit reporter OT-I mice with ROSA26-eYFP mice, were used. CD8 + T cells in these mice recognize the epitope SIINFEKL (OVA 257-264 ) and report active Hobit expression (by tdTomato expression) and previous Hobit expression (by YFP expression), which allows the detection of ex-Hobit + cells (ex-T RM ; YFP + tdTomato − ). First, we tested vaccination with OVA 257-264 -SLP in a prime-boost-boost setting, and this resulted in vaccine-specific CD8 + T cell responses that are comparable to other SLPs with respect to the kinetics and magnitude of the response (Fig. 5a) as well as the induction of the KLRG1 + CD62L − phenotype after the 1 st booster vaccination (Fig. 5b), which further corroborates that vaccine-specific conditions drive the differentiation of elicited CD8 + T cells.
Next, we adoptively transferred the OT-I CD8 + T cells of the Hobit LT mice into naïve wild-type mice that received subsequent OVA 257-264 -SLP vaccinations in a prime-boost-boost setting. As expected, an increase of CD8 + T RM cells (CD69 + YFP + tdTomato + ) was observed in the liver after the 2 nd and 3 rd vaccination, which were all co-expressing CD38 (Fig. 5c, d). Remarkably, in particular, the 3 rd vaccination induced the emergence of ex-T RM cells in the liver, spleen, and blood circulation, and the formation of these cells was characterized by the KLRG1 + CD44 + phenotype (Fig. 5d-g). Thus, ex-T RM cells with a KLRG1 + T EM phenotype are induced upon sequential SLP vaccination and are systemically present in the circulation.

Discussion
Here, we have investigated the capacity of single B cell and T cell epitope-containing peptide vaccines to elicit protection against SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model, and found that only a third vaccination with a long peptide harboring a single T cell epitope provided full protection. To our knowledge, this is the first study to show that vaccineelicited CD8 + T cells without the aid of virus-specific CD4 + helper T cells or neutralizing antibodies can protect against SARS-CoV-2, albeit provided in a booster setting requiring at least 2 boosters. These results may be of interest for the current discussion regarding a third vaccination, as the current vaccines elicit virus-specific T cells 4 . In addition, these results may also guide the development of T cell focused vaccines, which could be of utmost importance for susceptible patient groups such as transplantation and leukemia patients, and patients with autoimmune diseases treated with anti-CD20 antibodies such as rituximab. These patients have impaired antibody responses, and therefore may depend on robust T cell-eliciting vaccines for protection.
The DNA vaccine platform we used encoding Spike protein was highly efficient in generating neutralizing antibodies and resulted in protection against SARS-CoV-2 infection. Although, our results indicated that antibody responses to single linear B cell epitopes are inferior for the induction of neutralizing antibodies, we cannot exclude the possibility that single linear B cell epitopes exist that can elicit neutralizing antibodies. In addition, in settings in which antibodies mediate protection by other mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC), antibodydependent cellular phagocytosis (ADCP), and complementdependent cytotoxicity (CDC) 33 , the B cell-SLP platform may be valuable. In this respect, the addition of CpG and IFA as adjuvants and the insertion of a CD4 + T helper cell epitope to the linear B cell epitope vaccine was indispensable to elicit antibody responses.
In-depth studies using the T cell epitope SLP vaccines indicated that a third vaccination resulted in superior generation of CD8 + T EM cells, as exemplified by the expression of KLRG1, Ly6C, and the chemokine receptors CXCR3 and CX3CR1, in the circulation and also of CD8 + T RM cells in liver and lungs. The CD8 + T cells elicited upon a third dose vaccination are functionally and phenotypically modulated as evidenced by activation-associated cellsurface markers and their polyfunctional cytokine production. The latter is in line with a recent study in humans, which indicated that a third vaccination in kidney transplant recipients leads to increased circulating polyfunctional CD4 + T cells 34 . It would be of interest to decipher whether a third dose of the mRNA vaccine in healthy individuals, which is more effective against severe COVID-19-related outcomes compared to two doses 35 , also associates to increased T cell immunity. It is also of interest to examine whether differential time-intervals between the prime and booster vaccinations have impact on the magnitude and phenotype of the vaccine-elicited T cells 36,37 . A recent study indicated only differences for the induction of IL-2-expressing CD4 + T cell responses by mRNA vaccines when comparing intervals of 3-6 weeks to 8-16 weeks between the 1 st and 2 nd dose but shorter intervals were not compared 37 . Strikingly, the phenotype and cytokine polyfunctionality was highly similar between the different SLP vaccines, indicating that vaccine-specific conditions are dominant in shaping the T cell phenotype rather than the epitope used. This is in line with other studies showing that the environmental cues during T cell activation dictate the differentiation of these cells 31,38,39 .
The increase of T EM and T RM cells in the lungs after the third vaccination as reported here may be critical as the lungs are the primary entry point for SARS-CoV-2. Nevertheless, the circulating vaccine-induced T cells in the spleen and blood may also contribute to protection as well because these cells can rapidly migrate into the infected lung tissue and exert effector function. Moreover, also the efficient formation of the T RM cells in the liver, which was mainly observed after the third vaccination, may contribute to protection as these cells have superior potential to differentiate into ex-T RM cells 40 .
In conclusion, a third vaccination with a synthetic vaccine containing a single CD8 + T cell epitope results in protection against SARS-CoV-2 in the absence of neutralizing antibodies. This protection can be explained by an improved quantitative and qualitative CD8 + T cell response after the third vaccination that is highlighted by higher numbers of virus-specific T EM and T RM cells with polyfunctional cytokine capacity.
Committee (permit number AVD1160020186804), the Animal Tests Committee, and the Animal Welfare Body of the LUMC, and performed according to the recommendations and guidelines set by LUMC and by the Dutch Experiments on Animals Act.

SARS-CoV-2 infection.
Clinical isolate SARS-CoV-2/human/NLD/Leiden-0008/ 2020 (here named SARS-CoV-2) was used for the SARS-CoV-2 infection of mice. The next-generation sequencing data of this virus isolate is available under Gen-Bank accession number MT705206.1 and shows one mutation in the Leiden-0008 virus Spike protein compared to the Wuhan spike protein resulting in Asp>Gly substitution at position 614 (D614G). In addition, several non-silent (C12846U and C18928U) and silent mutations (C241U, C3037U, and C1448U) in other genes were found. Isolate Leiden-0008 was propagated and titrated in Vero-E6 cells (ATCC CRL-1586). K18-hACE2 transgenic mice were anaesthetized with isoflurane gas and intranasally infected with 5 × 10 3 plaque forming units (PFU) of SARS-CoV-2 in a total volume of 50 μl DMEM. Mouse weight and clinical discomfort were monitored daily. Euthanasia criteria were weight loss of >20 percent of body weight compared to the pre-study weight and a moribund state. All experiments with SARS-CoV-2 were performed in the Biosafety Level 3 (BSL3) Laboratories at the LUMC.
Antigen-binding ELISA. ELISAs were performed to determine antibody titers in sera. Nunc ELISA plates were coated with 1 µg/ml Spike S1 + S2ECD-His recombinant protein (SinoBiologicals, 40589-V08B1) in ELISA coating buffer (Biolegend) overnight at 4°C. Plates were washed five times and blocked with 1% bovine serum albumin (BSA) in PBS with 0.05% Tween-20 for 1 h at room temperature. Plates were washed and incubated with serial dilutions of mouse sera and incubated for 1 h at room temperature. Plates were again washed and then incubated with 1:4000 dilution of horse radish peroxidase (HRP) conjugated antimouse IgG secondary antibody (SouthernBiotech, cat. 1030-05) and incubated for 1 h at RT. To develop the plates, 50 µL of TMB 3,3=,5,5=tetramethylbenzidine) (Sigma-Aldrich) was added to each well and incubated for 5 min at room temperature. The reaction was stopped by the addition of 50 µL 1 M H 2 SO 4 , and within 5 min the plates were measured with a microplate reader (model 680; Bio-Rad) at 450 nm.
Single-cell preparations. Peripheral blood was collected from the tail vein. Splenocytes were obtained by mincing the tissue through cell strainers. Blood cells and splenocytes were depleted of erythrocytes using ammonium chloride lysis buffer. To remove remaining circulating blood cells from the liver and lungs, mice were perfused with 20 ml PBS containing 2 mM EDTA. Next, liver and lungs were cut into small pieces using surgical knives, and the tissue was resuspended in 3.5 ml or 1 ml, respectively, of IMDM containing 250 U/ml collagenase type 1-A (C2674, Sigma) and 20 μg/ml DNase I (D5025, Sigma). After incubation with collagenase/ DNase for 25 min at 37°C, liver and lung tissue was dissociated into single-cell suspensions using 70 μm cell strainers, and subsequently lymphocytes were isolated using a Percoll (GE Healthcare) gradient.
Flow cytometry. Following resuspension in staining buffer (PBS + 1% FCS) the cells were incubated with fluorescently labeled antibodies purchased from BioLegend, BD Biosciences, Invitrogen, or ThermoFisher (eBioscience). All antibodies used are listed in Supplementary Table 2 and in the Nature Research Reporting Summary linked to this article. Zombie Aqua (BioLegend) staining was used to exclude dead cells. To stain for EOMES and TCF1, cells were fixed for 30 min using the Fixation/Permeabilization concentrate (Invitrogen, FOXP3/Transcription factor Staining buffer set), diluted 1:6 in Fixation/Perm diluent (eBioscience). Following 2 washes in Permeabilization Buffer, anti-TCF1 and anti-EOMES antibodies (diluted in Permeabilization Buffer) were added and the cells were incubated for 1 h. Following a washing step in staining buffer the cells were ready for analysis. Glucose uptake was measured by the uptake of glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG, Life Technologies). Cell surface and intracellular cytokine stainings of splenocytes and blood lymphocytes were performed as described 42 . PADRE-specific CD4 + T cells were quantified using an MHC class II tetramer for the epitope (AKFVAAWTLKAA) (NIH Tetramer Core Facility). Spike 539-546 -specific, GP 34-41 -specific or OVA 257-264specific CD8 + T cells were quantified using MHC class I tetramers generated inhouse. For examination of intracellular cytokine production, single cell suspensions were stimulated with short peptides for 5 h in the presence of brefeldin A or with long peptides for 8 h of which the last 6 h in presence of brefeldin A (Golgiplug; BD Pharmingen). Flow cytometric acquisition was performed on a BD Fortessa flow cytometer (BD Biosciences) with BD FACS DIVA software (version 9) or on the Aurora Cytek spectral analyzer with SpectroFlo acquisition software (version 3), and samples were analyzed using FlowJo software (TreeStar) and OMIQ data analysis software (Gating strategy shown in Supplementary Fig. 5).
Adoptive T cell transfers. Spleens of OT-I Hobit reporter × ROSA26-eYFP LT mice were isolated and subsequently, OT-I CD8 + T cells were isolated by negative selection using MicroBeads (130-104-075, Miltenyi Biotec) according to the manufacturer's protocol. Splenic OT-I CD8 + T cells were adoptively transferred via retro-orbital injection into naïve mice.